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The use of body condition and haematology to detect
widespread threatening processes in sleepy lizards (Tiliqua
rugosa) in two agricultural environments
Anita K. Smyth, Elizabeth Smee, Stephanie S. Godfrey, Mathew Crowther and David
Phalen
Article citation details
R. Soc. open sci. 1: 140257.
http://dx.doi.org/10.1098/rsos.140257
Review timeline
Original submission:
Revised submission:
Final acceptance:
23 August 2014
20 October 2014
18 November 2014
Note: Reports are unedited and appear as
submitted by the referee. The review history
appears in chronological order.
Review History
RSOS-140257.R0 (Original submission)
Review form: Reviewer 1 (Michael Gardner)
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
I believe so
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
I do not feel qualified to assess the statistics
© 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use,
provided the original author and source are credited
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2
Recommendation
Accept with minor revision (please list in comments)
Comments to the Author(s)
The authors provide an interesting study which highlights that chemicals in the environment at
one site (the Severe site) have affected the health of sleepy lizards. Mostly the manuscript is well
written and appropriately analysed. I am some reservations regarding the use of a site from a
genetically different lineage to compare as a baseline but understand that this information may
not have been available to the researchers when they set up their study. I do have some
reservations about how the researchers have determined that the Baseline site was chemical free
as little information was provided here to back up these claims. I have a few suggestions to assist
in improving the manuscript. Abstract: Saying that the body size differences are due to genetic
differences is a bit premature. The differences could be genetic but also environmentally induced
– the study doesn't investigate this question specifically. Later in the discussion this argument is
more complete but still there may be many reasons for the body size difference which may be
reflective of environmental differences or an interaction between environment and genetics.
Page 3 Line 53-55: Change “…only a limited number of studies have been done examining the
value of…” to “…only a limited number of studies have examined the value of…” Page 4
Sentence starting line 5: It is unclear what multiple routes is referred to here – the different food
items eaten by herbivores, omnivores and carnivores would expose them to different routes of
chemicals also. I suggest rewording to have the multiple routes part of the sentences up front and
eg the different routes including herbivory etc. Page 4 Paragraph starting line 14: I think you need
to set up the idea that sleepy lizards are potentially good sentinel rather than start the sentence
saying they are. I understand that is a linking sentence so alternatively, perhaps this sentence
could start by saying that one species that fufills many of the criteria for being a good sentinel is
sleepy lizards. Then explain some of these attributes in the next sentence “For example….” Then
the paragraph should reworded to be about what makes the species a good sentinel rather than
being species rundown.
Page 4 Line 27/28: I think sleepy lizards breed in alternate years rather than annually, that
reference only covers the home rage estimates and not the annual breeding.
Page 5 line 45: What evidence is there to back up the statement “…the Baseline site were
considered to be exposed to fewer agricultural chemicals.”
Page 7 line 32: How was it possible to determine the age of the lizards? Do you mean age class
such as juvenile, sub adult and adult? More information is required here – what sizes were used
to establish “age”
Page 8 Line 9/10: Change “Total WBC counts were not done for samples collected from the
Severe sites. They were not done because the estimated WBC count is linked to the number of
RBCs.” To “Total WBC counts were not done for samples collected from the Severe sites because
the estimated WBC count is linked to the number of RBCs.”
Page 8 Line 37: spell out SVL at its first use, although familiar to lizard ecologists it might not be
to everyone. Page 8 Paragraph starting line 49: Were all tick life stages collected? B. limbatum is
Amblyomma limbatum – a different genus.
Page 9 line 45: At what site were the two dead lizards found?
Page 10 line 43: Two tick species are mention in the methods but the results don’t mention where
each was found. Can the prevalence of each species be presented in Table 1?
Page 11 line 56: Please define “H:L” as I believe this is the first time it has been mentioned. Page
15 sentence starting line 7: this sentence should appear before the previous sentence as it relates
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3
to the potential for differences due to genetic drift/selection rather than environmental
conditions which is the subject of the sentence immediately currently preceding it.
Page 15 line 11: insert “genetic” between “different” and “lineages”
Page 15 line 12: Misspelling of Gardner.
Page 15 line 13: I am not sure the argument that 3 generations of lizards were examined and
hence evidence that is valid. Firstly, how sure can the researches be that 3 generations were
sampled by Lancaster et al (which is essentially the same study). I don’t believe it is possible to
determine the age of these lizards directly. Secondly, if an environmental condition that has been
around for the time frame of 3 generations would potentially result in all generations being
smaller.
Page 16 line 29: Was the existence of the discharge recorded for individuals? If so was there any
relationship between the presence of discharge and any of the hematological measures?
Review form: Reviewer 2 (Reinier Mann)
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation
Accept with minor revision (please list in comments)
Comments to the Author(s)
This is a very interesting paper. As the authors state, reptile responses to chemical stresses have
only rarely been examined, and I applaud the authors for making a concerted attempt. I actually
have few criticisms of the manuscript as it stands, and my comments should be viewed largely as
suggestions that might improve the manuscript. Although the title of the paper is rather
circumspect as to the stresses at play within the cropland fields, the Introduction, Discussion and
indeed the Conclusion of the manuscript, are quick to implicate agricultural chemicals as a causal
factor in the poor body indices and anomalies in haematological parameters.
It would have been nice to see some information on the timing of pesticide/rodenticide
applications and the timing of lizard sample collections. It would also have been nice to see some
attempt to measure soil concentrations of some of these chemicals. In particular, chlorpyrifos
(spelt incorrectly on page 20, line26) and fibronil are likely to be persistent enough to show up in
soil analyses. It might also explain the absence of ectoparasites. Please note that the Portugal
study cited on page 20 has not actually been cited with the reference numbers [3, 4]. Also,
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4
although we did do a laboratory study with chlorpyrifos because we knew this chemical had
been used in the past in the those fields in Portugal, this chemical was not known to have been
used during our study, and we did not detect it in the soil in the cited studies.
It would be interesting to know if a lizard would actually eat a 10/80 bait. It seems to me that the
most likely candidate for poisoning is actually secondary poisoning by zinc phosphide (although
I would have thought its use was restricted to grain stores), and I had hoped that this would be
explored further - there is a reasonably extensive literature (see for example Bumbrah, G.S.,
Krishan, K., Kanchan, T., Sharma, M. and Sodhi, G.S. 2012. Phosphide poisoning: A review of
literature. Forensic Science International 214: 1-6). Did the timing of application of zinc phosphide
coincide with the collection of lizards - there is a statement on page 21 (line 31) that suggests that
lizards collected later were not affected by haematological anomalies.
On page 21 Both Aggarwal et al (1999) [46] and Bogle etal (2006) [47] have actually been misscited. Both these studies reported on the effects of aluminium phosphide (not zinc or aluminium
phosphate) in humans, not in reptiles. It is not clear which reptile study the authors may have
been referring to, but I don't think a study on zinc phosphate is likely to be relevant, because
phosphate is not the same as phosphide. I would have liked to see an explanation as to why the
baseline animals were collected from a site on, as the authors note, the other side of the river.
Would it not have been useful to collect a secondary (or primary) set of control animals from, for
example, the Billiatt Conservation Park?
On page 18 (line 43), the authors refer to “RBC membrane defects”; however this term had not
been referred to previously. Are they referring to the “circular cytoplasm defects” indicated in the
results? Those cytoplasmic vacuoles(?) do not seem to equate with the statements made on page
18, and the authors should clarify this. Finally, I would like to have seen at least some discussion
about the pros and cons of the non-invasive techniques, and how they might be complemented
by other, more invasive, biomarker studies, particularly those which may help elucidate the cause
of the haemolytic anaemia, or the likely involvement of the pesticides - particularly the
rodenticide. I’d be very interested to know if a lizard that eats a moribund mouse dying of
phosphine poisoning would display the same haematological parameters as you have seen
here............... Reinier M. Mann, PhD Editor, Australasian Bulletin of Ecotoxicology and
Environmental Chemistry http://ww2.setac.org/sapau/publications.html
Centre for Environmental Sustainability (CEnS), University of Technology, Sydney (UTS) Email:
[email protected] Water Assessment & Systems, Environmental Monitoring and
Assessment Sciences, Department of Science, Information Technology, Innovation and the Arts
(DSITIA) GPO Box 2454, Brisbane QLD 4001, Australia Ph: +61 7 3170 5559, Fax: +61 7 3170 5799
Decision letter
26-Sep-2014
Dear Dr Phalen,
The Subject Editor assigned to your paper ("USE OF BODY CONDITION AND
HAEMATOLOGY TO DETECT WIDESPREAD THREATENING PROCESSES IN SLEEPY
LIZARDS (TILIQUA RUGOSA) IN TWO AGRICULTURAL ENVIRONMENTS") has now
received comments from reviewers. We would like you to revise your paper in accordance with
the referee and Subject Editor suggestions which can be found below (not including confidential
reports to the Editor). Please note this decision does not guarantee eventual acceptance. Please
submit a copy of your revised paper within 14 days - if we do not hear from you within this time
then it will be assumed that the paper has been withdrawn. In exceptional circumstances,
extensions may be possible if agreed with the Editorial Office in advance. Once submitted your
paper may be returned to the previous referees, or new ones if these are unavailable.
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5
To revise your manuscript, log into http://mc.manuscriptcentral.com/bl and enter your Author
Centre, where you will find your manuscript title listed under "Manuscripts with Decisions."
Under "Actions," click on "Create a Revision." Your manuscript number has been appended to
denote a revision. Revise your manuscript and upload a new version through your Author
Centre. When submitting your revised manuscript, you must respond to the comments made by
the referees and upload a file "Response to Referees" in "Section 6 - File Upload". Please use this
to document how you have responded to the comments, and the adjustments you have made. In
order to expedite the processing of the revised manuscript, please be as specific as possible in
your response.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look
forward to receiving your revision. If you have any questions at all, please do not hesitate to get
in touch.
Yours sincerely,
Charlotte Wray
Senior Publishing Editor,
Royal Society Open Science
[email protected]
Author's Response to Decision Letter for (RSOS-140257)
See Appendix A.
RSOS-140257.R1 (Revision)
Review form: Reviewer 2 (Reinier Mann)
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No.
Recommendation
Accept with minor revision (please list in comments)
Comments to the Author(s)
The authors have addressed all the comments and concerns of the two reviewers.
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A
from http://rsos.royalsocietypublishing.org/ on June 15, 2017
Dear Editor,
We appreciate the comments by the reviewers and have considered them carefully. We
have addressed each of these below and feel that these changes have improved our paper.
We hope that you will now find this paper acceptable for publication.
Sincerely yours,
David Phalen
Comments to the Author(s)
This is a very interesting paper. As the authors state, reptile responses to chemical stresses have
only rarely been examined, and I applaud the authors for making a concerted attempt. I actually
have few criticisms of the manuscript as it stands, and my comments should be viewed largely as
suggestions that might improve the manuscript.
Although the title of the paper is rather circumspect as to the stresses at play within the cropland
fields, the Introduction, Discussion and indeed the Conclusion of the manuscript, are quick to
implicate agricultural chemicals as a causal factor in the poor body indices and anomalies in
haematological parameters.
It would have been nice to see some information on the timing of pesticide/rodenticide
applications and the timing of lizard sample collections.
Au: The following has been added to address the issue of when various agricultural
chemicals were applied and to discuss the implications of this timing.
Page 19 line 24 However, neither the frequency and timing (potentially anytime of the year)
of baiting
Page 20, line 1. Resource Management, South Australia) nor the use of Paraquat®
(April/March) has varied.
Page 21, line 4 - 9.
On the other hand, the timing of the application of organophosphates and fipronil argues
against them being the cause of the anaemia. Organophosphate application was only done
from the beginning of October until the end of November. Anaemic animals and animals
with polychromasia were found in September before the spraying occurred and therefore
the only way that these chemicals could have been contributory would if the lizards were
exposed to residual concentrations from the previous year.
Page 21, line 13-17.
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Most application would have occurred in March and April when the cereal crops were
sown, but could have been applied in the winter and then again in the spring as well (K.
Haebich, Team Leader, Rangelands and Rivers NRM Group, Department of Environment
and Resource Management, South Australia).
Page 22 line 1 – line 10
Sleepy lizards enter brumation soon after grain crops are sown. Studies on wound healing
suggest that reptiles at temperatures below their prefered optimum temperature zone have
a reduced metabolic rate and healing is delayed [48]. It would also be likely that animals in
brumation would not be able to respond to a toxic insult during brumation and signs of
exposure might persist until the following spring. We therefore consider it possible and
even likely that sleepy lizards poisoned in the autumn may have still been anaemic in the
spring as they emerge from brumation and that additional application of zinc phosphide in
the spring could have resulted in finding toxic changes during the entire study period. In
the end, however, because of the exposure to multiple potentially toxic chemicals, the cause
of the haemolytic anaemia seen in these sleepy lizards will require controlled exposure
experiments before it can be determined.
It would also have been nice to see some attempt to measure soil concentrations of some of these
chemicals. In particular, chlorpyrifos (spelt incorrectly on page 20, line26 Au: corrected) and
fibronil are likely to be persistent enough to show up in soil analyses. It might also explain the
absence of ectoparasites.
Au: The work was done sufficiently long enough ago that testing soil at this time would not
be meaningful. Future studies will include soil testing.
Please note that the Portugal study cited on page 20 has not actually been cited with the reference
numbers [3, 4]. Au: Corrected. Also, although we did do a laboratory study with chlorpyrifos
because we knew this chemical had been used in the past in the those fields in Portugal, this
chemical was not known to have been used during our study, and we did not detect it in the soil
in the cited studies.
It would be interesting to know if a lizard would actually eat a 10/80 bait.
Au: For future research. This will require experimental trials and an animal ethics
approval.
It seems to me that the most likely candidate for poisoning is actually secondary poisoning by
zinc phosphide (although I would have thought its use was restricted to grain stores), and I had
hoped that this would be explored further - there is a reasonably extensive literature (see for
example Bumbrah, G.S., Krishan, K., Kanchan, T., Sharma, M. and Sodhi, G.S. 2012. Phosphide
poisoning: A review of literature. Forensic Science International 214: 1-6).
Au: Page 21 line 18-23: The following has been added to the text. Zinc phosphide is
rapidly converted to phosphine gas when it is exposed to hydrochloric acid in the stomach.
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It causes cell death in many organs as the result of disruption of energy metabolism in the
mitochondria. It effects RBCs by oxyhaemoglobin to methaemoglobin and thus could have
resulted in the damaged RBCs seen in the sleepy lizards and the resultant haemolytic
anaemia [47].
Did the timing of application of zinc phosphide coincide with the collection of lizards - there is a
statement on page 21 (line 31) that suggests that lizards collected later were not affected by
haematological anomalies.
Au: Please see comments inserted above.
On page 21 Both Aggarwal et al (1999) [46] and Bogle etal (2006) [47] have actually been misscited. Both these studies reported on the effects of aluminium phosphide (not zinc or aluminium
phosphate) in humans, not in reptiles. It is not clear which reptile study the authors may have
been referring to, but I don't think a study on zinc phosphate is likely to be relevant, because
phosphate is not the same as phosphide.
Au: Deleted
I would have liked to see an explanation as to why the baseline animals were collected from a
site on, as the authors note, the other side of the river. Would it not have been useful to collect a
secondary (or primary) set of control animals from, for example, the Billiatt Conservation Park?
Au: Retrospectively this might have been a good choice. However, at the time the
experiment was designed Mt. Mary’s was chosen because of the long history of study at this
site.
On page 18 (line 43), the authors refer to “RBC membrane defects”; however this term had not
been referred to previously. Are they referring to the “circular cytoplasm defects” indicated in
the results? Those cytoplasmic vacuoles(?) do not seem to equate with the statements made on
page 18, and the authors should clarify this.
Au: We never used the word vacuoles. A vacuole is a membrane lined structure. We feel
that these are actual holes in the cytoplasm which is a defect.
Finally, I would like to have seen at least some discussion about the pros and cons of the noninvasive techniques, and how they might be complemented by other, more invasive, biomarker
studies, particularly those which may help elucidate the cause of the haemolytic anaemia, or the
likely involvement of the pesticides - particularly the rodenticide. I’d be very interested to know
if a lizard that eats a moribund mouse dying of phosphine poisoning would display the same
haematological parameters as you have seen here...............
Au. The next step would be to do experimental toxin exposure trials and we state in the
paper that there is a need for controlled exposure trials. We did not do invasive testing and
cannot comment on how it would compare to invasive testing. We think it would be
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assumed that if an exposure experiment was done, the animals would be euthanized and
extensive testing of the tissues would be done.
Reviewer: 2
Comments to the Author(s)
The authors provide an interesting study which highlights that chemicals in the environment at
one site (the Severe site) have affected the health of sleepy lizards. Mostly the manuscript is well
written and appropriately analysed. I am some reservations regarding the use of a site from a
genetically different lineage to compare as a baseline but understand that this information may
not have been available to the researchers when they set up their study.
Au: This information was not available at the time of the study. Nor was the
morphometric data that suggested that these animals might have been genetically different.
I do have some reservations about how the researchers have determined that the Baseline site
was chemical free as little information was provided here to back up these claims.
Au: We have provided as much information that is available regarding the chemicals
applied to both sites.
I have a few suggestions to assist in improving the manuscript.
Abstract: Saying that the body size differences are due to genetic differences is a bit premature.
The differences could be genetic but also environmentally induced – the study doesn't investigate
this question specifically. Later in the discussion this argument is more complete but still there
may be many reasons for the body size difference which may be reflective of environmental
differences or an interaction between environment and genetics.
Au: We say that our findings combined with those that have shown that these two
populations are genetically different suggest that the difference in size could be caused by
genetic differences. We feel this is a sufficiently cautious statement that it will not be over
interpreted.
Page 3 Line 53-55: Change “…only a limited number of studies have been done examining the
value of…” to “…only a limited number of studies have examined the value of…”
Au: change made.
Page 4 Sentence starting line 5: It is unclear what multiple routes is referred to here – the
different food items eaten by herbivores, omnivores and carnivores would expose them to
different routes of chemicals also. I suggest rewording to have the multiple routes part of the
sentences up front and eg the different routes including herbivory etc.
AU: Page 3, line 20: (inhalation, ingestion, and direct skin contact) added after routes.
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Page 4 Paragraph starting line 14: I think you need to set up the idea that sleepy lizards are
potentially good sentinel rather than start the sentence saying they are. I understand that is a
linking sentence so alternatively, perhaps this sentence could start by saying that one species that
fufills many of the criteria for being a good sentinel is sleepy lizards. Then explain some of these
attributes in the next sentence “For example….”
Then the paragraph should reworded to be about what makes the species a good sentinel rather
than being species rundown.
Au: The paragraph begins by saying the sleepy lizards has the potential to be … We do
not see a need to change the way that it is written.
Page 4 Line 27/28: I think sleepy lizards breed in alternate years rather than annually, that
reference only covers the home rage estimates and not the annual breeding.
Au: Page 4 line 5–7 added: Young are born mostly annually (assuming pairings lead to
breeding success) [16 and references therein] and remain within the same home range as
their mothers during the first year [17].
Page 5 line 45: What evidence is there to back up the statement “…the Baseline site were
considered to be exposed to fewer agricultural chemicals.”
Au: We have addressed this in detail in the discussion – (see discussion) has been added
after this statement.
Page 7 line 32: How was it possible to determine the age of the lizards? Do you mean age class
such as juvenile, sub adult and adult? More information is required here – what sizes were used
to establish “age”
Au: This is an omission due to version control during editing. The lizards were grouped
into juveniles (SVL<200 mm) and adults which included two sub-adults (SVL>200 mm).
Reworded the text to reflect this clarification.
Page 8 Line 9/10: Change “Total WBC counts were not done for samples collected from the
Severe sites. They were not done because the estimated WBC count is linked to the number of
RBCs.” To “Total WBC counts were not done for samples collected from the Severe sites
because the estimated WBC count is linked to the number of RBCs.”
Au: Done
Page 8 Line 37: spell out SVL at its first use, although familiar to lizard ecologists it might not
be to everyone.
Au: Not required now as spelt out on Page 7, line 32 above.
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Page 8 Paragraph starting line 49: Were all tick life stages collected? B. limbatum is
Amblyomma limbatum – a different genus.
Au: We’ve reworded the text to reflect that all tick life stages were collected but we didn’t
grouped by species or life stage in this study.
Corrected B. limbatum to Amblyomma limbatum.
Page 9 line 45: At what site were the two dead lizards found?
Au: Added – in the Severe site
Page 10 line 43: Two tick species are mention in the methods but the results don’t mention
where each was found. Can the prevalence of each species be presented in Table 1?
Au: We didn’t collect data on tick counts by species as we used the total number of feeding
ticks to indicate blood loss because we reasonably assumed the variation in feeding
quantities would be affected by season, life stage, species and lizard habitat and cooccurring hosts would be similar and the overall statistical error associated with pooling
the error would be low. Our results indicating that tick load in the Severe didn’t
consistently influence blood loss supports this. However, in the Baseline sites where tick
load (sheer number) was influential and interspecific differences in blood intake should be
considered when investigating further the influences of feeding ticks on lizard physiological
health. It would be a mandatory filter in a study of that type where was the main research
focus is tick feeding but that wasn’t our research focus. Another related is using the ticks
collected in our study for PhD study (Flinders University Australia) which is yet to be
published and submitted for examination.
Page 11 line 56: Please define “H:L” as I believe this is the first time it has been mentioned.
Au: Changed to read - heterophil to lymphocyte rations (H:L)
Page 15 sentence starting line 7: this sentence should appear before the previous sentence as it
relates to the potential for differences due to genetic drift/selection rather than environmental
conditions which is the subject of the sentence immediately currently preceding it.
Au: Changes made
Page 15 line 11: insert “genetic” between “different” and “lineages”
Au: Added
Page 15 line 12: Misspelling of Gardner.
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Au: Corrected
Page 15 line 13: I am not sure the argument that 3 generations of lizards were examined and
hence evidence that is valid. Firstly, how sure can the researches be that 3 generations were
sampled by Lancaster et al (which is essentially the same study). I don’t believe it is possible to
determine the age of these lizards directly. Secondly, if an environmental condition that has been
around for the time frame of 3 generations would potentially result in all generations being
smaller.
Au: This bit of text that should have been omitted and was overlooked during our editing.
It has been removed.
Page 16 line 29: Was the existence of the discharge recorded for individuals? If so was there any
relationship between the presence of discharge and any of the hematological measures?
Au: Unfortunately this data was not recorded. Page 16 line 15 and 16 the following has
been added: A correlation between WBC and observed signs in these animals would have
been ideal, but was not recorded.